Interaction of peptide substrates of fibroblast collagenase with divalent cations: Ca2+ binding by substrate as a suggested recognition signal for collagenase action.

To correlate structural data on substrates of human fibroblast collagenase with their interaction with the enzyme, we have studied: Ac-PLG-s-LLG-O-ethyl ester (I), Dnp-PLGLWA(d-Arg)-NH2 (II), AcGPEGLRVG-O-ethyl ester (III) and Succ-GPLGP-O-amidomethylcoumaryl ester (IV). Peptides I and II represent collagenase cleavage sequences in collagen, peptide III is a mimic for the cleavage site in alpha 2-macroglobulin and peptide IV represents a non-substrate model. Kinetic data showed that peptides I, II and III were substrates of the enzyme. In contrast, peptide IV was not acted upon by the enzyme. Circular dichroism data on the peptides showed that the peptides assume ordered structures in water and trifluoroethanol. In the latter solvent, peptides I and III bound Ca2+ and Zn2+ while peptide II bound Ca2+ but not Zn2+. Peptide IV did not bind either cation in this solvent. Together with the kinetic data, the results suggest that the collagenase cleavage segments in collagen and non-collagen substrates of collagenase could interact with Ca2+ and the enzyme to form a ternary complex. This, in turn, would imply a cofactor role for Ca2+ in collagenase action in addition to the solely structural role ascribed so far to this cation.